The effects of flaxseed and tamoxifen on the inflammatory microenvironment in normal breast tissue and in breast cancer

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The effects of

flaxseed and tamoxifen

on the inflammatory

microenvironment

in normal breast tissue

and in breast cancer

Linköping University Medical Dissertation No. 1714, 2019

GABRIEL LINDAHL

GA BR IE L L IN DA HL 20 19 Th e e ffe cts o f fl ax se ed a nd t am ox ife n o n t he i nfl am m ato ry m icr oe nv iro nm en t i n n orm al b re as t t iss ue a nd i n b re as t c an ce r

Linköping University medical dissertations, No. 1714 ISBN: 978-91-7929-963-7

ISSN: 0345-0082

FACULTY OF MEDICINE AND HEALTH SCIENCES (IKE) Linköping University

SE-581 83 Linköping, Sweden www.liu.se

”Barn’s burnt down

Now

I can see the moon”

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The effects of flaxseed and tamoxifen

on the inflammatory microenvironment

in normal breast tissue and in breast cancer

Gabriel Lindahl

Division of Surgery, Orthopedics and Oncology Department of Clinical and Experimental Medicine Linköping University, SE-581 85 Linköping, Sweden

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2 © Gabriel Lindahl, 2019

Cover: Linum Usitatissimum, Magnus Petersson, 2019.

ISBN: 978-91-7929-963-7 ISSN: 0345-0082

Published articles have been reprinted with the permission of the copyright holders: Paper I 2011, American Association for Cancer Research

Paper II 2019, Springer Nature Paper III 2019, Frontiers Media SA

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Abstract

Breast cancer is the most common cancer among women worldwide today. Nearly 9000 women are diagnosed with breast cancer in Sweden yearly and despite advantages in

diagnostics and treatments approximately 1400 women still die from their disease every year. Breast cancer has a diverse etiology and hormonal factors and life-style factors contribute to an increased breast cancer risk. High mammographic density is also considered a risk factor but the underlying mechanisms are not fully understood. Inflammation is associated with poor survival in several malignancies and is considered a hallmark of cancer. There is evidence indicating that increased inflammation is associated with dense breast tissue and may contribute to an increased risk of breast cancer in these patients.

There is an urgent need to find risk reduction strategies in breast cancer prevention. Several studies have shown that antiestrogens significantly reduce breast cancer incidence in women with high risk of developing breast cancer and can be used for chemoprevention. These drugs may have potentially severe side effects and other strategies are needed. Dietary interventions may influence breast cancer risk without any major side effects. Studies indicate that dietary phytoestrogens may reduce breast cancer risk. The most common phytoestrogens in Western populations are lignans, mainly found in flaxseed, but results from several studies with lignans for breast cancer prevention have been inconsistent.

In this thesis we investigated the effects of tamoxifen and flaxseed on inflammatory mediators in normal breast tissue and in breast cancer. We used the microdialysis technique to sample proteins from the extracellular space in vivo. This technique gives us the opportunity to study proteins in their bioactive compartment in situ and to study changes in protein levels at different time points without affecting the tissue of interest. We also used experimental models and cell cultures to study tumor growth of human breast cancer xenografts, cancer cell proliferation and angiogenesis.

In paper I, we investigated whether tamoxifen, flaxseed, enterolactone or genestein reduced growth of human breast cancer xenografts and their association with pro-inflammatory cytokine interleukin 1β (IL-1β) and its antagonist interleukin 1 receptor antagonist (IL-1Ra). In paper II, we investigated whether tamoxifen and flaxseed exerted similar effects on inflammatory mediators in normal breast tissue in vivo. In paper III, we investigated whether osteopontin (OPN), a pro-inflammatory cytokine, was associated with dense breast tissue and breast cancer and if tamoxifen and flaxseed could alter OPN levels in normal breast tissue in vivo. We also investigated the correlation between OPN and inflammatory mediators in normal breast tissue and in breast cancer in vivo.

In conclusion, we showed that tamoxifen and flaxseed affected breast cancer growth in an experimental model and may exert an anti-inflammatory effect in breast cancer and normal breast tissue by increasing the IL-1Ra/IL-1β ratio in vivo. We showed that dense breast tissue and breast cancer were associated with increased levels of OPN. Circulating estrogen did not correlate to OPN and tamoxifen and flaxseed did not affect OPN levels suggesting an estrogen independent regulation of OPN in vivo. These finding contributes to our understanding of how tamoxifen and flaxseed affects inflammation and the role of inflammation in the pathogenesis of breast cancer.

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Populärvetenskaplig sammanfattning

Bröstcancer är den vanligast förkommande cancersjukdom hos kvinnor idag. I Sverige insjuknar närmare 9000 kvinnor i bröstcancer varje år varav cirka 1400 kommer att dö till följd av sin sjukdom.

Det finns flera bakomliggande orsaker till att kvinnor drabbas av bröstcancer såsom

hormonella faktorer och levnadsvanor. En ökad täthet i bröstvävnaden på mammografibilder, ”täta bröst”, har också kopplats till en ökad risk att drabbas av bröstcancer. Den

bakomliggande mekanismen mellan täta bröst och bröstcancer är inte helt klarlagd, men det finns studier som talar för att en ökad inflammation i bröstvävnaden kan vara en förklaring. Inflammation är en känd riskfaktor för att utveckla vissa typer av cancer och inflammation är förknippad med en sämre prognos vid flera cancersjukdomar inklusive bröstcancer.

Det är viktigt att försöka hitta sätt att förebygga bröstcancer. Flera studier har visat att förebyggande medicinering med läkemedel som blockerar östrogen, antiöstrogen, och som idag används för att behandla bröstcancer, är effektiva för att skydda mot insjuknande av bröstcancer, men att de finns en risk för allvarliga biverkningar såsom livmoderkroppscancer och blodproppar med dessa läkemedel. Studier har visat att kosten möjligen kan påverka risken för att insjukna i bröstcancer och att växtöstrogener eventuellt kan ha en skyddande effekt. Det vanligaste förekommande växtöstrogenet i kosten i västvärlden är lignaner som finns i olika sädesslag, bär, nötter och frön, och då framför allt i linfrön.

Vi har undersökt hur tamoxifen, ett antiöstrogen, och linfrö påverkar tillväxten av

bröstcancertumörer hos möss och vilka biologiska mekanismer som påverkar tillväxten med fokus på inflammation. Vi har även studerat hur olika proteinernivåer förändras i

bröstcancertumörer och i normal bröstvävnad hos kvinnor efter behandling med tamoxifen eller ett dagligt tillskott av 25 g preparerade linfrön i kosten under sex veckor. Slutligen har vi också undersökt kvinnor med täta bröst och jämfört nivåerna av ett inflammationsprotein i bröstvävnaden med kvinnor med bröstcancer och kvinnor med icke-tät bröstvävnad. För att kunna undersöka protein i bröstvävnaden har vi använt en metod som kallas för mikrodialys. Den går i korthet ut på att man för in en mycket tunn kateter genom huden och in i

bröstvävnaden. I katetern flödar en vätska som drar till sig ämnen i vävnaden och som på så vis kan samlas upp och analyseras.

Våra resultat visar att både tamoxifen och linfrö bromsar tillväxten av mänsklig bröstcancer i ett experiment på möss och att vi får en minskning av ett viktigt inflammationsprotein, interleukin 1 beta (IL-1β), och en ökning av ett protein som hämmar IL-1βs funktion,

interleukin 1 receptor antagonist (IL-1Ra), och som sannolikt bidrar till den positiva effekten. Vi kunde också visa att tamoxifen och ett kosttillskott med linfrö hade en liknande sannolikt inflammationsdämpande effekt med en ökning av IL-1Ra i bröstvävnad hos friska kvinnor. Slutligen kunde vi visa att kvinnor med täta bröst och bröstcancer hade en likartad förhöjd nivå av ett annat viktigt inflammationsprotein, osteopontin (OPN) jämfört med icke-tät bröstvävnad. Däremot verkade nivåerna av OPN inte påverkas av behandling med tamoxifen eller ett kosttillskott av linfrö.

Sammanfattningsvis talar våra resultat för att tamoxifen eller ett kosttillskott av linfrö kan ha positiva effekter med en möjlig dämpning av vissa inflammationsproteiner i bröstvävnad. Vi

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har också visat att täta bröst och bröstcancertumörer har ökade nivåer av ett viktigt

inflammationsprotein, OPN, och som möjligen kan förklara den ökade risken för bröstcancer som finns hos kvinnor med täta bröst, men att detta protein inte påverkas av tamoxifen eller ett kosttillskott av linfrö. Dessa resultat kan ge en ökad förståelse för kopplingen mellan inflammation och bröstcancer och den eventuella bröstcancerförebyggande effekten av linfrö.

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List of scientific papers

This thesis is based on the following papers:

1. G. Lindahl, N. Saarinen, A. Abrahamsson, C. Dabrosin, Tamoxifen, flaxseed, and the lignan enterolactone increase stroma- and cancer cell-derived IL-1Ra and decrease tumor angiogenesis in estrogen-dependent breast cancer. Cancer Res 71, 51-60 (2011). 2. G. Lindahl, A. Abrahamsson, C. Dabrosin, Dietary flaxseed and tamoxifen affect the

inflammatory microenvironment in vivo in normal human breast tissue of postmenopausal women. Eur J Clin Nutr, (2019).

3. G. Lindahl, A. Rzepecka, C. Dabrosin, Increased Extracellular Osteopontin Levels in Normal Human Breast Tissue at High Risk of Developing Cancer and Its Association With Inflammatory Biomarkers in situ. Front Oncol 9, 746 (2019).

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Abbreviations

AIs aromatase inhibitors

BI-RADS Breast Imaging Reporting and Data System

CAFs cancer associated fibroblasts

CCL chemokine (c-c motif) ligand

CKs chemokines

COX-2 cyclooxygenase 2

CRP c-reactive protein

CTLs cytotoxic T cells

CXCL chemokine (c-x-c motif) ligand

DC dendritic cells

DNA deoxyribonucleic acid

E2 estradiol

ECM extra cellular matrix

ELISA enzyme-linked immunosorbent assay

ENL enterolactone

ER estrogen receptor

GEN genistein

HRT hormone replacement therapy

HUVECs human umbilical vein endothelial cells

IFNγ interferon gamma

IHC immunohistochemistry

IL-18BP interleukin 18 binding protein

IL-1R interleukin 1 receptor type

IL-1Ra interleukin 1 receptor antagonist

IL-6RA interleukin 6 receptor subtype alpha

ILs interleukins

MBAA multiplex bead array assay

MCF-7 Michigan Cancer Foundation-7

MD mammographic density

MMPs matrix metalloproteases

MVD microvessel density

NF-κB nuclear factor kappa-light-chain-enhancer of activated B cells NK cells natural killer cells

NPX normalised protein expression

NSAIDs nonsteroidal anti-inflammatory drugs

OPN osteopontin

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PCR polymerase chain reaction

PEA proximity extension assay

s.c. subcutaneous

SDG secoisolariciresinol diglucoside

SERM selective estrogen receptor modulator

SHBG sex hormone binding globulin

sST2 soluble suppressor of tumorigenicity 2

Tam tamoxifen

TAMs tumor associated macrophages

Th cells helper T cells

TIMPs tissue inhibitors of matrix metalloproteinases

TME tumor microenvironment

TNF tumor necrosis factor

Treg regulatory T cells

uPA urokinase Plasminogen activator

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Introduction

Breast cancer is one of the oldest diseases documented throughout history. In an early Egyptian papyrus manuscript, possibly dated 1600 BC, several cases of breast cancer are described and concluded to be incurable (1). Endocrine treatment for breast cancer was introduced by Dr George Thomas Beatson, who performed a bilateral oophorectomy on a woman with recurrent breast cancer in 1895. However, the treatment was combined with sheep thyroid extract and the link between oophorectomy and breast cancer was purely empirical, and the mechanisms of action unknown. In 1897, an English surgeon, Dr Stanley Boyd, wrote a paper where he put forward the hypothesis that, “internal secretion of the ovaries in some cases favors the growth of cancer”. Years later he published his summary data and concluded that one third of his breast cancer patients benefited from the procedure although no patients were cured (2). In the late 19:th century, Dr William S. Halstead introduced a new surgical technique to treat breast cancer with a curative intent by removing the breast en bloc with the underlying pectoralis muscle and axillary lymph nodes (3). Since then, the surgical techniques have been refined and other treatment modalities have been introduced such as radiotherapy in the 1930s, adjuvant chemo- and hormonal therapy in the 70s, and since the turn of the millennium, new targeted drugs. Furthermore, in the 70s and 80s screening mammography was introduced throughout the Western world, including Sweden, and pooled estimates from several randomized control trials have shown a reduction in breast cancer mortality by at least 20 % due to screening (4). Taken together breast cancer mortality rate in the European Union has declined since the peak rate in 1989 of more than 20 /100,000 to an estimated 13.4 / 100,000 in 2020 (5). Still, breast cancer is the most common cancer among women worldwide and despite recent advantages in diagnostics and treatment, the leading cause of cancer-related death (6). In Sweden, close to 9000 women will be diagnosed with breast cancer annually, and despite a decrease in mortality rate during the last 30 years, approximately 1400 women will die from breast cancer every year (7).

The etiology of breast cancer is diverse, but long term exposure to sex steroids and genetic alterations are well-known risk factor. Estrogens play an important role in the development and progression of breast cancer and approximately 80% of all breast cancers are estrogen-receptor (ER) positive (8). The use of antiestrogens such as selective estrogens-estrogen-receptor modulator (SERMs) and aromatase inhibitors (AIs) are gold standard in the treatment of ER-positive breast cancer today. SERMs are also approved as chemoprevention to women at high risk of breast cancer in several countries such as USA, Canada and the United Kingdom, but may have potentially severe side effects (9, 10).

However, other contributors such as life-style factors and chronic inflammation may also be possible causes of breast cancer. These conclusions come from migrant studies showing an increased breast cancer risk in women who move from countries with low incidence to countries with high incidence, and epidemiological studies indicating that women who regularly use anti-inflammatory drugs may have a lower breast cancer risk (11, 12). Inflammation is considered to be a hallmark of cancer and the tumor microenvironment has become more and more recognized as a critical player in all steps of carcinogenesis (13). Stromal cells and immune cells have been shown to produce a variety of growth factors, cytokines and proteases creating an inflammatory milieu stimulating angiogenesis and tumor

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proliferation. Furthermore, inflammation has been identified as a negative prognostic factor in several malignancies including breast cancer (14, 15).

Dietary habits may also affect the risk of breast cancer. Phytoestrogens are plant-derived dietary compounds that interacts with the estrogen receptor and therefore potentially can affect all processes regulated by estrogens (16). In a Western population, the most common phytoestrogens are lignans found mainly in flaxseed. Studies on lignan intake and breast cancer risk have however been inconclusive, where some studies have shown no association and others an inverse association in subpopulations of women, e.g., the postmenopausal (17, 18). To conclude, breast cancer incidence and mortality is the highest of all female

malignancies despite advances in diagnostics and treatment. There is a need to further understand the underlying mechanisms of this disease and to explore possible preventive strategies. In this thesis we wanted to explore how phytoestrogens and tamoxifen (Tam) affect inflammation in breast tissue and breast cancer, hopefully elucidating a small part of this enigmatic disease. After all, every single step counts when you embark on a journey of a thousand miles.

Breast cancer and risk factors

The breast consists of different compartments, such as exocrine glands, ducts and supporting stromal and fat tissue. The breast undergoes continuous changes during a woman’s different reproductive phases and reaches full maturity and function during pregnancy and lactation (19). The maintenance and differentiation of mammary gland cells is dependent on signaling from the local microenvironment by growth factors and cytokines. These interactions influence the changes that occur during development, pregnancy and lactation but also during tumorigenesis (20, 21).

Breast cancer is basically defined as the presence of a malignant tumor that originates from epithelial cells in the glands or ducts of the breast. The tumor cells proliferate and acquire the ability to invade surrounding tissues, lymph nodes and distant organs. Breast cancer is a heterogeneous disease that originates from a normal precursor cell following an accumulation of oncogenetic changes (22). It has been shown that breast cancer cells accumulate up to 1000s of mutations, but despite this multitude of mutations, breast cancers can phenotypically be divided into four major subgroups with similar clinical behavior (23). Apart from genetic changes, preclinical studies indicate that the microenvironment also affects the tumor cells and promotes or inhibits further neoplastic transformation (24). The microenvironment’s role in cancer progression is furthermore illustrated by the fact that more than one third of women aged 40-50 have in situ breast cancer in autopsy studies, yet breast cancer is diagnosed in only 1% in this age group (25). This suggests that the tumor remains dormant, and that additional events in the stroma are needed for tumors to develop into clinical cancer.

The incidence of breast cancer has steadily increased during the last century and the lifetime risk of developing female breast cancer is today over 10% in the Western world. Age, reproductive history, hormonal exposure, obesity, life style factors such as high alcohol consumption and low physical activity, precancerous breast lesions, chest radiation, family history and, mutations in predisposition genes are all associated with an increased risk of

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developing breast cancer (26). Mutations in predisposition genes, such as mutations in BRCA1 and BRCA2, are by far the strongest risk factor but account for only approximately 2-3 % of all breast cancer, whereas lifestyle factors, reproductive history and hormonal exposure, such as hormone replacement therapy (HRT), have a limited but significant impact on the risk increase (27).

Breast density

On mammography, epithelial cells and connective tissue attenuates x-rays more compared to surrounding adipose tissue and appears radiologically dense. Several factors such as genetics, parity, menopausal status, HRT, and diet can influence mammographic density (MD), and approximately 10 % of all women in the US are considered to have an extremely high MD corresponding to a MD ≥75% of a mammogram (28). High MD is a risk factor for breast cancer in both pre- and postmenopausal women and women with extremely high MD have a four- to fivefold greater risk of breast cancer compared to women with low MD categorized as <5% or <10% in different studies (29, 30). There are several biological factors associated with differences in MD. Biopsies have shown more epithelial cells and/or stromal tissue, and especially a greater proportion of collagen. One plausible explanation is thus that MD correlates with epithelial tissue at risk of malignant transformation but also to a larger stromal component potentially containing inflammatory cells and mediators (31). Studies have investigated the association between inflammatory markers in breast tissue sections and MD with inconsistent results (32, 33). However, we have shown that postmenopausal women with extremely high MD have a more pro-inflammatory microenvironment compared to women with low MD which could contribute to a higher risk of developing breast cancer (34). Furthermore, we have also compared postmenopausal women with dense breast tissue to women with breast cancer and found that 26 of 32 inflammatory mediators showed similar profile (35).

There is limited knowledge of the association between MD and survival in women diagnosed with breast cancer. A recent meta-analysis indicated that high MD at diagnosis was associated with an increased risk of recurrence and higher mortality, and that a reduction in MD during breast cancer treatment is a positive predictive factor. However, due to mostly retrospective studies and different assessment methods the results have to be interpreted carefully (36).

Estrogens and the estrogen receptors

Estrogens are steroid hormones derived from cholesterol by the successive action of steroidogenic enzymes in the ovaries and through peripheral conversion of circulating precursors, androgens, in peripheral tissues (Figure 1). In women, the level of estrogen synthesis is high during the reproductive years to later decline during the transition and postmenopausal period. Estrogen exists in three forms of which 17β-estradiol (E2) is the most abundant and potent form (37). Estrogens are crucial in reproduction and normal

physiological processes in both males and females, but are also associated with pathological conditions such as metabolic disorders, dementia, cardiovascular disease, osteoporosis and cancer (38). There is compelling evidence that estrogens play a major role in the development

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15 Figure 1. Simplified overview of estrogen metabolism.

Cholesterol is converted to androgens which are subsequently aromatized to estrogens. Estrone is reversibly converted to estradiol.

Blue ellipses: converting enzymes. 17β-HSD: 17β-hydroxysteroid dehydrogenases.

of breast cancer and that high lifetime exposure increases the risk of breast cancer.

Oophorectomized women have a significantly reduced breast cancer risk, as do women with a late onset of menarche and early menopaus, and in postmenopausal women increased levels of circulating estrogens are associated with a higher risk of breast cancer (39). Furthermore, approximately 80% of all postmenopausal breast cancers are ER-positive and respond to antiestrogen treatment (8).

E2 is a small liposoluble molecule mainly synthezised in the ovaries and released into the general circulation in premenopausal women. In postmenopausal women E2 is produced by conversion of androgens by aromatase enzymes in breast, brain and fat tissue where it acts locally. E2 passively enters the cell through the plasma membrane and its actions are mainly mediated by its binding to the ER; alpha (ERα), beta (ERβ) or any of their isoforms, localized mainly in the nucleus. When activated by their ligand the receptor dimerize and activate or repress gene transcription by binding to specific deoxyribonucleic acid (DNA) sequences in the genome (40).

Both ER subtypes are expressed in various tissues and while ERα primarily is expressed in female reproductive organs, ERβ is mainly expressed in non-gonadal tissues (41). The expression of ERα is scarce in normal breast tissue but becomes highly upregulated in breast cancer, where it has been shown to promote tumorigenesis and progression of the disease, while the expression of ERβ is decreased and even lost during breast cancer progression. Furthermore, the proliferation of ERα-positive breast cancer cells is enhanced by estrogens, which induce multiple growth factors, cyclins and cytokines involved in cell survival and cell cycle progression (38). The role of ERβ in breast cancer has been debated, but several in vitro studies indicate a protective role of ERβ through inhibited proliferation and increased apoptosis (42) . ERβ protective role is also supported by a meta-analysis that showed an association between ERβ expression and improved disease free survival independent of ERα status (43).

The concentration of circulating E2 is also dependent on the concentration of sex hormone-binding globulin (SHBG), a serum protein synthesized in the liver in response to hormonal and non-hormonal factors such as physical activity and diet (44). SHBG regulates the bioavailable fraction of E2 and studies have shown an inverse association between SHBG and postmenopausal breast cancer risk (45).

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Antiestrogens

SERMs are molecules that bind to the ER with the capacity to exert different effects on various estrogen-related targets. Tam is the most common SERM and has been used in clinical practice for treatment of breast cancer since the mid-70s. It is metabolized by various cytochrome P450 enzymes to more active metabolites that competitively binds to both ERα and ERβ and partly attenuates and inactivates downstream gene transcription (46). Tam produces different effects depending on the target tissue and exerts antagonist activity in breast but agonist activity in bone and the uterus. (47). Due to estrogens many physiological functions in different tissue such as reproductive organs, bone and cardiovascular system, there have been concerns on how to minimize possible adverse effects from these tissues (48). Tam is widely used in the treatment of ER-positive breast cancer both in the adjuvant and metastatic setting. The use of Tam as adjuvant treatment for 5 years has shown a reduction in absolute breast cancer mortality rates by 9 percentage points (24% vs 33%) in ER-positive breast cancer after 15 years, but with no benefit for patients with ER-negative cancer. However, the use of Tam also results in a slightly increased risk of endometrial cancer in the elderly population (49). Other SERMs, such as raloxifen, have been developed but none has yet shown any significant advantage over Tam. 20-30% of ER-positive breast cancers develop resistance to Tam. This mechanism is not well understood, but upregulation of membrane bound ERs may contribute to Tam resistance (50).

Another class of anti-estrogens are selective estrogen downregulators, such as fulvestrant. Fulvestrant binds to the ER but without any downstream activation of transcript factors and with a rapid degradation of the receptor and is considered a pure antagonist (48). Fulvestrant is today mainly used in treatment of metastatic breast cancer after progression of previous endocrine therapy (51).

Another endocrine approach for breast cancer treatment is to deprive ER of E2. This was previously done by oophorectomy and/or adrenalectomy but nowadays the same effect is accomplished in postmenopausal breast cancer patients with AIs that inhibit estrogen synthesis in peripheral tissue by blocking the aromatase enzyme. Several studies have shown that AIs are superior to SERMs in the treatment of postmenopausal women and is now considered gold standard in the adjuvant and metastatic setting (52, 53).

As chemoprevention in women with an increased risk of breast cancer, SERMs have been tested and approved and has shown to reduce the risk of invasive breast cancer almost by half, but with an increased risk of endometrial cancer and thromboembolic events (54, 55). AIs have also shown to be efficient in chemoprevention, and with fewer side effects than SERMs (56). However, they are not yet approved for chemoprevention by medical authorities as there are concerns regarding long term adverse effects on bone loss and cardiovascular risk.

Phytoestrogens

Phytoestrogens are plant-derived dietary compounds that represent a diverse group of naturally occurring chemicals characterized by structural similarity to E2. They bind to ERs and can potentially affect all the processes regulated by estrogens, but exert a weaker estrogenic activity than E2 (16, 57). Phytoestrogens can be divided into different groups of

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which the most common are isoflavones, such as genistein (GEN) or daidzein, mostly found in soy products but also in grapes and fruits, and lignans, mainly enterolactone (ENL) and enterodiol, found in seeds, whole grains, vegetables and fruits (Figure 2) (58, 59).

Dietary habits may affect the risk of breast cancer and studies of migrant populations of Asian immigrants in USA show a higher, and increasing, incidence in breast cancer compared to their native counterparts. The increasing incidence is probably due to lifestyle factors including, among others, decreased consumption of soy products (11). There are also studies indicating that the timing of exposure to soy/isoflavones is important and that exposure early in life, i.e., before menarche, leads to a greater risk reduction of breast cancer (60) .

Dietary phytoestrogen may be a non-toxic prevention strategy to reduce the incidence of breast cancer, but the evidence is not irrefutable and phytoestrogen consumption is not incorporated in the World Cancer Research Funds (WCRF) breast cancer prevention recommendations (61). Furthermore, phytoestrogens might induce potential adverse health effects since they may act as endocrine disruptors and negatively affect fertility in women (62).

Enterolactone

In Western populations lignans are the most common type of phytoestrogen found in high concentrations in seeds, fruits and vegetables (63). Flaxseed is the richest source of lignans consumed by humans and contains the lignan secoisolariciresinol diglucoside (SDG) (59).

SDG must be converted by intestinal bacteria to its active metabolites, mainly ENL, to exert a biological effect (16, 64). The metabolism of enterolignans includes conjugation, first-pass metabolism and enterohepatic recirculation both in the bowel mucosa as well as in the liver (65). Due to a high individual difference in gut microbial diversity and composition there is

Figure 2. Comparison of the chemical structures of

estrogen 17β-estradiol with phytoestrogens: Isoflavones, genistein and daidzein, Lignans, enterolactone and enterodiol.

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an individual difference in the bioavailability of dietary enterolignans (63, 66). Previous use of antibiotics may also reduce gut microbiota which impair uptake of lignans. To estimate individual lignan consumption and metabolism, serum or urinary levels of enterolignans can be measured (67).

Phytoestrogens and breast cancer

Phytoestrogens seem to preferentially interact with ERβ and may influence the risk of breast cancer by inhibiting cell proliferation, but also by initiating apoptotic events and inhibiting angiogenesis presumably in an ER-independent manner (16, 68). There might, however, be a biphasic effect of phytoesterogens exerting a stimulatory effect at low doses, but acting as an antiestrogen at higher doses suppressing cell growth. This has been shown in breast cancer cell culture where low doses of ENL induced a stimulatory effect while high doses had an inhibitory effect on cell growth in vitro (69). Moreover, low doses of isoflavones have shown to induce breast cancer cell proliferation through the stimulation of ERα in mouse models (70, 71). In an estrogen depleted mouse model with human breast cancer xenografts, a dietary addition of flaxseed showed no increased tumor growth compared to unexposed mice, and contrarily, an addition of dietary soy showed tumor progression (72). Phytoestrogens different effects on breast cancer growth are also supported by other models where the dietary addition of flaxseed or ENL, but not GEN, inhibited tumor growth, presumably by a vascular

endothelial growth factor (VEGF)-mediated effect (73-75).

The mechanisms of action of phytoestrogens are thus not fully understood and may not only include interaction with the ER. Studies have shown that phytoestrogens suppress the transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), alter the expression of proteins that control the cell cycle and apoptosis in breast cancer cell lines, and interfere with estrogen converting enzymes (68). Furthermore, flaxseed contains fatty acids, such as omega-3, which may have anticancer properties (76). An experimental model showed that flaxseed oil attenuated breast cancer growth and reduced cancer cell proliferation (77). ENL has also been shown to stimulate the production of SHBG which binds free E2 and may result in lower concentrations of circulating sex hormones thereby potentially decreasing breast cancer cell growth (78). There may also be an anti-inflammatory effect of ENL in breast cancer, as a retrospective study showed an inverse association between ENL, c-reactive protein (CRP) and mortality (79). However, an earlier meta-analysis of 20 prospective randomized studies of various conditions and diseases, including prostate cancer but not breast cancer, showed no effect of flaxseed on reducing CRP (80).

The effects of phytoestrogens on risk reduction in breast cancer have to some extent been inconclusive. Whereas a recent meta-analysis of several prospective studies of consumption of isoflavonoid containing foods and breast cancer risk suggested a reduced risk with high consumption of soy products, results from epidemiological studies on the association between lignans and/or ENL and breast cancer are more uncertain (17, 18, 67, 81, 82). In a review article from 2007 the author concludes that out of ten prospective and 18 case-control studies published, no or almost no significant associations between plasma ENL or lignan intake and breast cancer risk were reported (81). Since then, results from a large Swedish prospective

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cohort study has shown a significant inverse association between lignan intake and postmenopausal breast cancer risk, especially among women using hormone replacement therapy (18). A protective effect of lignans was also demonstrated in a large case-control study and a meta-analysis showing a significant inverse association between serum levels of ENL and risk of postmenopausal breast cancer, especially in the ER-negative population (67). Conversely, a large European prospective study showed no association between lignan intake and reduced risk of breast cancer, but a significant decrease in breast cancer mortality in postmenopausal women (17, 83). Other studies support the association between high

concentrations of serum ENL and reduced mortality in postmenopausal breast cancer patients (78, 84). However, the results of epidemiological trials must be interpreted cautiously since there may be limitations in the included studies, such as different self-reported questionnaires to assess exposure, different cut-off values for categorizing the study population and lack of information about important confounding factors e.g., exposure to hormone replacement therapy.

Phytoestrogens does not seem to be associated with breast density; a study of MD in postmenopausal women showed an association between high levels of ENL and a slightly increased percentage MD but of uncertain clinical significance and a recent meta-analysis showed no association between isoflavones and MD in postmenopausal women (85, 86)

The tumor microenvironment and inflammation

In 1863 the German pathologist Rudolf Virchow described leukocyte infiltration in neoplastic tissues and suggested that cancer may originate from chronic inflammation. Since then the concept of inflammation as a key event in carcinogenesis has become fully accepted and today it is estimated that approximately 25 % of all cancers are associated with chronic inflammation (87).

In 2000 Hanahan and Weinberg published a paper arguing that carcinogenesis is a multistep process of stochastic events leading to malignant transformation. They described tumor-acquired capabilities shared by most human tumors, the hallmarks of cancer, and categorized these into six categories; self-sufficiency in growth signals, insensitivity to anti-growth signals, tissue invasion and metastasis, limitless replicative potential, sustained angiogenesis and evading apoptosis (88). In 2011 they revised their model in the light of recent advances in cancer research and added two hallmarks, avoiding immune destruction and deregulating cellular energetics, but they also introduced a new concept of tumor initiating properties; genomic instability and mutation, and tumor-promoting inflammation (13).

Tumor development has been compared to a non-healing wound as they share many similarities such as angiogenesis, influx of inflammatory cells and remodeling of the stroma (89). Thus, the tumor microenvironment (TME) has become increasingly recognized for its collaborative interaction with tumor cells in all steps of carcinogenesis. Cancer associated fibroblasts (CAFs) are an important component of the cancer stroma. They are heterogeneous in origin, but seem mainly to be converted from resident fibroblasts by tumor released cytokines (90). CAFs have been described to play a major role in remodelling of the extracellular matrix (ECM) by expressing matrix metalloproteases (MMPs), stimulate

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angiogenesis by upregulating VEGF and promote tumor proliferation via secretion of various cytokines such as tumor necrosis factor (TNF), interleukin (IL) 1 beta (IL-1β), IL-6,

osteopontin (OPN) and chemokine (c-x-c motif) ligand (CXCL) 12 (90, 91). Collagen is the main protein of the ECM and is important for the structure of the tissue as well as facilitating intracellular communication and studies have demonstrated the importance of CAFs and ECM-proteins within the stroma for cancer initiation, growth and metastatic spread (92-94).

Inflammation and the immune system

The immune system consists of two major effector pathways; the immediate non-specific innate immune response and the subsequent specialized adaptive immune response. The innate immune system includes several hematopoetic cells such as macrophages, dendritic cells (DCs), neutrophils, mastcells and natural killer (NK) cells. In addition, epithelial cells lining the surfaces of the body participate in the innate immune response together with circulating inflammatory proteins such as CRP. The innate immune response recognizes and eliminates external pathogens expressing ‘non-self’ molecules and unhealthy cells with upregulated abnormal ‘induced self’ molecules due to infection, stress and malignancies (95). The dysregulation of transcriptional factors, such as NF-κB, is a key event in cancer related inflammation and immunity. NF-κB may be constitutively activated in cancer cells or immune cells by genetic alterations, but is mainly activated by external factors such as hypoxia and/or pro-inflammatory cytokines, e.g., IL-1 and TNF. NF-κB stimulates several genes, which play a major role in cell survival, resistance to cell death, migration and angiogenesis (Figure 3) (91, 96). Moreover, these transcription factors coordinate the production of inflammatory mediators including MMPs, ILs and chemokines (CKs), and as a consequence inflammatory leukocytes are recruited to the TME further sustaining the inflammation (97).

Figure 3. NF-κB and the hallmarks of cancer.

Hallmarks and inflammatory mediators discussed in this thesis shown in black.

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Macrophages are derived from monocytes in peripheral blood and constitute a major component of the inflammatory infiltrate. They can be activated by two distinct pathways where classically activated macrophages (M1) following exposure to interferon gamma (IFNγ) and microbial products are tumoricidal, whereas alternative activated macrophages (M2) following exposure to cytokines, such as IL-4, IL-10, chemokine (c-c motif) ligand (CCL) 2 and CCL17 and 22, are considered immunoregulatory and cancer promoting (96). During cancer initiation, macrophages are characterized as more anti-tumoral (M1), but as the tumor secretes cytokines in the TME, such as IL-6, IL-10 and IL-33, the macrophages change into a more pro-tumoral M2 phenotype (98). This effect may be due to physiological changes in the TME, such as pronounced hypoxia and pH-changes, during cancer progression (96) However, the polarization of macrophages is dynamic and not absolute, resulting in a functional polymorphism of TAMs (14).

As the cells of the innate immune system neutralize tumor cells initially, they activate the adaptive immune response by presenting tumor-associated antigens to T cells. This stimulates the proliferation of cytotoxic T cells (CTLs) and augmenting T helper cells which can eliminate tumor cells and activate B-cells (Figure 4). However, TAMs and DCs in the TME may attenuate the adaptive response by recruiting immunosuppressive regulator T cells (Treg) that suppress the anti-tumoral activity of other immune cells. Furthermore, the tumor cells evade elimination by expressing transmembrane proteins, programmed death-ligands, that deactivate effector CTLs (99). The role of B-cells in the TME is not fully understood. They may act anti-tumoral by differentiating into plasma cells and produce anti-tumor antibodies but further studies are necessary (100).

Figure 4. Cells of the innate and adaptive immune system in the TME

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Inflammation and breast cancer

The abundance of TAMs are associated with worse overall survival in breast cancer patients and the absence of TAMs have shown a significantly reduced invasiveness and metastatic potential in an experimental breast cancer model (15, 101). Conversely, other effector cells of the immune system, such as CTLs and NK cells, may eliminate breast cancer cells and are associated with better prognosis in ER-negative breast cancer, whereas infiltrating lymphocytes are associated with reduced survival in ER-positive breast cancer (102, 103). Inflammatory cytokines may induce a more aggressive phenotype of breast cancer and elevated levels of circulating acute-phase proteins, CRP and serum amyloid A, have been found to be prognostic markers for reduced overall survival in disease free breast cancer patients after adjuvant treatment, suggesting an association between inflammation and poor prognosis (103, 104).

In addition, several experimental studies of anti-inflammatory drugs, such as nonsteroidal anti-inflammatory drugs (NSAIDs), have shown reduced xenograft tumor growth in mouse models. NSAIDs induce their effects mainly by the inhibition of the enzyme cyclooxygenase (COX) 2, with subsequent reduction of COX-2 related pro-inflammatory metabolites. This may explain why NSAIDs may have a positive effect in breast cancer prevention, as COX-2 activities have been identified in various steps of breast carcinogenesis (105). The use of NSAIDs have however shown conflicting results in human studies. A large randomized trial with aspirin vs placebo in healthy women from 2005 showed no significant risk reduction of cancer in general or breast cancer in particular (106). However, a meta-analysis from 2008 showed that NSAID use was associated with reduced breast cancer risk and a more recent meta-analysis from 2015 showed a weak but significant dose-response relationship between acetylsalicylic acid and reduced breast cancer risk in premenopausal women (12, 107).

Inflammatory mediators - cytokines

The Interleukin 1 family of cytokines

Cytokines are highly localized messenger proteins which activate and mediate the immune response during inflammation. There is no clear distinction between different groups of cytokines such as ILs and CKs and several cytokines function as both. Traditionally, ILs have been recognized as mediators between immune cells and CKs as chemotactic factors. One of the central regulators of innate immunity and inflammation is the IL-1 family of cytokines (IL-1s). They may act as alarmins, or danger signals, released as a result of injury to initiate a local inflammatory response, which under normal physiological conditions leads to tissue regeneration, but when dysregulated can cause pathologic inflammation (108). The IL-1s comprises eleven cytokines divided into three subgroups; the IL-1 subfamily, the

interleukin 18 (IL-18) subfamily and the interleukin 36 (IL-36) subfamily (Table 1). Furthermore, there are ten members of the IL-1 family of receptor, including

anti-inflammatory receptors and decoy receptors such as IL-1 receptor type 2 (IL-1R2) and soluble suppressor of tumorigenicity 2 (sST2) (109). The main function of IL-1s is to initiate pro-inflammatory reactions in response to tissue injury by activation of NK-κB and subsequent expression of several other cytokines such as IL-6 and IL-8 (110). The two major IL-1

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agonistic molecules are IL-1α and IL-1β. IL-1α is constitutively expressed intracellularly, mainly in luminal epithelial cells, and though rarely secreted; it acts as an alarmin in response to tissue injury and initiates the innate immune system (109). IL-1β is primarily produced by monocytes, macrophages and DCs in response to complement components, cytokines or by autocrine signaling. IL-1β is synthesized as an inactive precursor in the cytoplasm and is activated through cleavage by caspase-1 before secretion into the extracellular space. IL-1β is a potent mediator of inflammation and thus highly upregulated in inflammatory and malignant tissue (111).

IL-1s are also known mediators of angiogenesis. In wound healing, this effect seems to be mediated through induction of hypoxia-inducible factor-1α and upregulation of VEGF, whereas in malignancies this seems mainly to be an effect mediated by activated NF-κB in TAMs and the expression of IL-6, IL-8 and adhesion molecules stimulating pro-inflammatory endothelial cells (112). Moreover, in experimental models, inhibition of IL-1β has shown greatly reduced angiogenesis (113, 114).

IL-18 is constitutively expressed by several cells including immune cells. Its main function is to modulate the immune response by activating NK cells and to enhance the adaptive immune response. Anti-tumoral effects of IL-18 have been demonstrated in animal models of early stages of inflammatory malignancies such as gastric and colon cancer. However, tumor promoting effects have been shown in advanced gastric cancer, melanoma and pancreas cancer (115). IL-18 binding protein (IL-18BP) is a natural inhibitor of IL-18 and is produced by monocytes and macrophages. In preclinical melanoma and lung cancer models,

administration of IL-18BP inhibited metastasis, and thus inhibition of IL-18 may have positive effects in tumors expressing IL-18 receptors (115).

The IL-1 subfamily also includes IL-33 which is expressed mainly by various non-immune cells and acts as an alarmin triggered by allergens or parasites. IL- 33 acts pro-inflammatory by inducing T cells differentiation and activating mast cells, but may also induce Tregs and thus attenuate the immune response (116). Studies have shown an overexpression of IL-33 in airway inflammatory diseases, inflammatory bowel disease and in several cancers such as colorectal, breast and lung cancer (116, 117). IL-33 is regulated by sST2, an extracellular decoy receptor that binds to IL-33 thereby preventing IL-33 signaling. Circulating sST2 is

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increased in intestinal and metabolic diseases and in breast cancer and, has shown to be useful as a biomarker in heart failure (118).

The IL-1 family includes a naturally occurring receptor antagonist (IL-1Ra) which antagonizes the activities of IL-1α and IL-1β by binding to IL-1R1 with higher affinity but without eliciting a cellular response (111). In preclinical studies, treatment with IL-1Ra has led to reduced angiogenesis and inhibited tumor development. A recombinant human IL-1Ra, anakinra, used for treatment of rheumatoid arthritis, is tested in early clinical trials on cancer patients and the addition of anakinra to chemotherapy in heavily treated patients with metastatic colorectal cancer has shown promising activity (119, 120).

The Interleukin 1 family of cytokines and breast cancer

IL-1β is elevated in several malignancies, including breast cancer, where it has been associated with higher tumor grade, aggressiveness, angiogenesis and poor prognosis (108, 121). Its importance in the carcinogenic process of breast cancer has been shown in several experimental studies where caspase-1 knockout mice with decreased IL-1β secretion, or mice treated with anakinra, have exhibited impaired tumor development and fewer metastases in lungs and bones. (122-124). There is data suggesting that mainly TAMs and DCs are associated with upregulated IL-1β expression and that high level of IL-1s, including IL-1β and IL-1Ra, in breast cancer tissue are associated with more advanced stage and worse outcome (124, 125). IL-1β enhances the activity of estrogen producing enzymes such as aromatase and steroid sulfatase in breast cancer cell culture, presumably stimulating the production of bioactive estrogens, which may suggest an estrogen mediated effect of IL-1β on breast cancer proliferation (126). This possible effect is furthermore supported by a positive correlation between E2 and IL-1β, and a negative correlation between E2 and IL-1Ra, in human breast tissue (127). In a small study of 11 metastatic breast cancer patients, treatment with anakinra in combination with chemotherapy eliminated a “signature of IL-1 associated inflammation” in blood cells suggesting an enhanced anti-tumor activity of anakinra (124). IL-18s role in breast cancer is not fully understood, but there is growing evidence that high serum levels of IL-18 are associated with worse outcome in breast cancer patients (128, 129). Serum levels of both IL-33 and sST2 have shown to be increased in patients with breast cancer and correlate to VEGF and MMPs (117). Furthermore, in a breast cancer model, IL-33 was associated with an immunosuppressive TME, accelerated tumor growth, increased angiogenesis and metastasis (130).

Interleukin 6

During acute inflammation IL-6 is mainly produced by monocytes and macrophages as a response to tissue damage and contributes to the recruitment of inflammatory cells (131). However, IL-6 is a pleiotropic cytokine with both pro- and anti-inflammatory properties. These properties are in part regulated by IL-1β which suppresses the anti-inflammatory effects of IL-6 (132). In cancer, IL-6 is secreted by both tumor and stromal cells and is known to induce a pro-tumoral M2 phenotype of macrophages and to affect several hallmarks of

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cancer, e.g., proliferation, angiogenesis, invasiveness and metastasis (98, 131). Elevated levels in serum and tissue have been observed in several malignancies and are generally associated with aggressive tumors and poor prognosis (133). IL-6 may also protect cancer cells from therapy induced damage (134). Treatment with IL-6 and IL-6 receptor inhibitors are currently being tested in several clinical cancer trials (135).

Interleukin 6 and breast cancer

The role of IL-6 in breast cancer is ambiguous. In epidemiological studies no significant correlation between circulating IL-6 and risk of developing breast cancer has been demonstrated (136). However, in breast cancer patients, high levels of serum IL-6 are associated with increased levels of VEGF and, higher tumor stage, higher metastatic burden and worse prognosis (137, 138). Conversely, expression of IL-6 within breast cancer tumors has shown an association with early stage and better overall survival (139). In cell culture, IL-6 has shown either inhibitory or promoting effects on tumor cell proliferation, in part

dependent on ER-status, whereas mouse models have shown an IL-6 dependent tumor growth and metastasis (140).

Interleukin 8

Endothelial cells secret IL-8 in both acute and chronic inflammation. IL-8 acts as a chemotactic factor for neutrophils and granulocytes and may promote angiogenesis by stimulating endothelial cell production of VEGF (141). IL-8 expression has been shown to be significantly higher in several types of malignancies and in vivo cancer models have shown that tumor derived IL-8 contributes to an immunosuppressive microenvironment and angiogenesis which promote tumor progression (142, 143).

Interleukin 8 and breast cancer

Studies support an estrogen-mediated effect of the regulation of IL-8 and we have previously demonstrated that E2 increased and Tam decreased IL-8 levels and that serum estrogen levels correspond to IL-8 levels in breast cancer tissue (144). Furthermore, serum levels of IL-8 correlates with more advanced disease and worse prognosis in patients with breast cancer (145, 146) . Currently, the use of IL-8 neutralizing antibodies and receptor antagonists are being tested in preclinical and early clinical trials for malignant diseases but the results have so far been disappointing (142).

Osteopontin

OPN was first described by Oldberg and co-workers in Lund in 1986 as a bone specific sialoprotein isolated from a rat osteosarcoma (147). They identified an extracellular protein that facilitated the interaction between cells and minerals in the matrix suggesting a role in bone development and remodeling. Later studies showed that OPN also is present in human epithelial lining cells communicating with the external environment implicating that OPN

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may have a protective role in the interaction between these surfaces and the environment (148).

Since then has OPN been identified in various tissues such as brain, liver, lung, breast, gastrointestinal tract, cardiac and kidney and can be detected in a variety of body secretions including urine, saliva and milk (149). However, OPN levels are low in healthy tissue but is upregulated in inflammation and wound healing where OPN is a key cytokine expressed by numerous cells including bone, muscle and endothelial cells contributing to the migration and activation of inflammatory cells (150). OPN is furthermore expressed by DCs, macrophages and activated T cells which enhance the inflammatory response (151).

There are both an intracellular and a secreted isoform of OPN of which the intracellular protein has been contributed to cellular processes in DCs related to migration, fusion and motility, whereas the extracellular isoform is thought to regulate the interaction with different target cells (152). Secreted OPN is subject to splicing as well as posttranslational

modifications, such as proteolytic cleavage by MMPs, which results in a functional diversity (153, 154) Pro-inflammatory cytokines, such as IL-1β, IL-10 and TNF, are reported to upregulate OPN secretion from DCs, while conversely IFNγ limits OPN expression (155). OPN is a central mediator in inflammation and regulates the immune system at different levels. It acts as a chemotactic factor for inflammatory cells such as macrophages,

neutrophils, DCs and NK cells (154). This has been shown in mouse models where the use of OPN-deficient mice and blocking antibodies greatly reduced the infiltration of macrophages and neutrophils (151, 156). OPN also plays an important role in the induction of the adaptive immune response and mediates T-cells differentiation important in the defense against bacterial infections (157). OPN is upregulate and associated with several autoimmune diseases such as multiple sclerosis, systemic lupus erythematosus and chronic inflammatory disease such as inflammatory bowel disease and atherosclerosis (158).

OPN is produced by several different cell types in the TME, but there is limited knowledge on whether tumor-derived and stroma-derived OPN differs functionally. In the TME OPN has been shown to activate TAMs essential for promotion of tumor growth, remodeling of tissues, and suppression of antitumor immunity (98). OPN also influence angiogenesis by inducing VEGF expression in endothelial cells (159, 160). Emerging data also support OPN as a key mediator in epithelial-mesenchymal transition necessary for cancer progression and metastasis through the stimulation of CAFs and the secretion of transforming growth factor and IL-6 (161, 162).

Several meta-analyses in different malignancies such as colorectal, breast, lung, prostate cancer and studies of melanoma and glioblastoma have shown reduced overall survival for patients with high levels of OPN in plasma or serum, or increased expression in tumor tissue (163-167). Phytoestrogens may influence OPN secretion and in preclinical studies of prostate cancer, GEN has shown a biphasic dose-dependent effect on OPN expression and OPN-mediated tumor growth and survival (168, 169).

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Osteopontin and breast cancer

In pre-malignant breast lesions, one study have shown that strong IHC staining of OPN-c, an isoform of OPN, is associated with a higher risk of malignant transformation and worse outcome (170). In breast cancer patients the results have been unclear. In a cohort of ER-positive early adjuvant breast cancer patients, neither OPN expression nor plasma levels were associated with reduced overall survival (171). However, a meta-analysis exploring OPN expression by immunohistochemistry (IHC) staining in breast cancer has shown that high expression of OPN is correlated with poor overall survival (166). Another study showed a negative association between OPN gene expression and overall survival in patients with breast cancer, and elevated and increasing plasma levels of OPN have also been associated with poor survival in metastatic breast cancer patients (172, 173). Furthermore, an in vivo model has shown that an overexpression of OPN increases lymphovascular invasion, lymph node metastases and lung micrometastases supporting OPNs role in breast cancer invasion and metastasis (174).

Chemokines

Chemotactic cytokines constitute a large family of structurally related small proteins characterized by their ability to stimulate cell migration. Today about 50 CKs, divided into four subgroups, and more than 20 receptors have been identified including decoy receptors, expressed mainly on erythrocytes, that do note elicit a response (175, 176). Membrane bound CKs form a chemotactic gradient required for directional cell migration and CK-activated receptors interact with integrins in the basement membrane resulting in a trans-endothelial migration of leukocytes. Furthermore CKs can act in synergy with other CKs or cytokines to elicit a more pronounced response. Some CKs are constitutively expressed and contribute to the maintenance of the immune system whereas inflammatory CKs are inducible and regulate leukocyte migration in response to infiltrating pathogens. Apart from their role in the

inflammatory response, CKs are also critical for guiding migrating cells during

embryogenesis. Nearly all tissues express CKs, but in normal breast tissue the expression is generally low (177, 178).

In tumors, both cancer and stromal cells produce CKs that contribute to both pro-tumoral activities, such as recruitment of TAMs and neutrophils, as well as anti-tumoral activities, such as recruitment of T lymphocytes and NK cells. CKs may also regulate angiogenesis and mediate metastasis (175).

There are several pro-tumoral CKs, like CXCL1-3, 5-7 and CCL2 and 5, that attract neutrophils and TAMs, promote angiogenesis and may play a role in chemotaxis and metastasis of tumor cells. CXCL12 play a distinct role in the metastatic process by attracting CK-receptor CXCR4-expressing tumor cells to distant sites. CXCL12 is released in large amounts by lung, liver, lymph nodes and bone marrow (179).

Other CKs seem to have pleiotropic effects; CXCL9-11 have anti-tumoral effects on the tumor microenvironment, like skewing TAMs to an anti-tumoral M1 phenotype, but may also have pro-tumoral effects on the tumor cells. The effects seem to depend on the CK-receptor

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phenotype expression and synergistic effects of other CKs (176). CXCL4 and CXCL14 have angiostatic and anti-tumoral effects and is downregulated in cancer tissue compared to adjacent normal tissue (180). Antibodies blocking different CK-receptors, e.g., CXCR4, have shown impaired tumor growth in animal models and are being tested in different clinical trials (181).

Chemokines and breast cancer

In an analysis of gene-expression of CKs in breast cancer, 27 CKs were upregulated in breast cancer tissue compared to adjacent normal breast tissue and nine of these CKs also showed elevated levels in plasma compared to healthy controls (182). In breast cancer, CCL2 and CCL5 is highly expressed in both tumor and stromal cells and are associated with high tumor grade and poor prognosis (183). In an animal model, the levels of CCL2 and CCL5 correlated with the levels of E2, indicating an estrogen dependent mechanism. Furthermore, treatment with Tam decreased in vivo levels of CCL2 and CCL5 in breast tissue (184). CXCR4 expression is upregulated in patients with breast cancer and high levels have been associated with regional and distant metastasis and reduced survival (179).

Inflammatory mediators - proteases

Matrix metalloproteases

MMPs were first identified as enzymes capable of hydrolysis of collagen and were originally associated with cancer invasion due to their ability to degrade the extracellular matrix facilitating cell migration (185). Later studies have shown that MMPs also cleave non-matrix substrates contributing to the release and modification of signaling molecules (186). As such, MMPs lead to the activation of several pro-inflammatory cytokines such as TNF and IL-1β as well as regulating chemokine gradients (187). However, the function of MMPs is often context-dependent and MMPs may also induce an anti-inflammatory response by proteolytic processing of inflammatory regulators (188).

To date 23 MMPs have been identified and most MMPs are secreted in an inactive form, subsequently activated in the extracellular space by several proteinases including plasmin and other MMPs (189). MMPs play a role in normal physiological events, such as tissue

homeostasis and wound healing, but generally the expression of MMPs is low in healthy tissue. Overexpression of MMPs can induce severe tissue damage and MMP hyperactivity has been observed in many pathological conditions, such as osteoarthritis, multiple sclerosis, and cancer (187).

In cancer MMPs have been described to promote proliferation, angiogenesis and ECM degradation facilitating tumor growth, invasion and metastasis (190). However, some MMPs, such as MMP-8, seem to have a protective effect on tumor invasion and metastasis and others, such as MMP-12 and MMP-9, may exert different effects dependent on if they are secreted by tumor cells or cells in the tumor stroma such as TAMs (191). The function of MMPs is highly regulated by four tissue inhibitors of matrix metalloproteinases (TIMPs) that strongly block the activity of MMPs. Dysregulation of TIMPs have been identified in most cancers indicating their importance in MMP regulation and tumor progression. Furthermore,

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decreased TIMP 3 expression has been correlated with advanced disease and poor prognosis in several cancers (192). Despite MMPs significant role in cancer, clinical trials with MMP-inhibitors in cancer treatment have all failed to reach their end points and have induced significant side effects. The poor efficacy was likely due to broad-spectrum MMP inhibition including cancer-protecting MMPs. Since these early trials, our understanding of MMPs diverse biological effects has deepened and, consequently, future development of more selective inhibitors might give better results with fewer adverse events (193).

Matrix metalloproteases and breast cancer

MMP gene expression in breast cancer has shown an association between MMP-9,-11 and -15 and poor survival (194). Furthermore, IHC-staining of MMP-2 and MMP-9 have shown significantly higher expression in breast cancer than in the surrounding breast tissue and positive staining correlates with higher tumor stage and poorer prognosis (195). MMP overexpression, especially MMP-9, have been associated with worse prognosis in several studies, but the results diverge concerning MMP-2 and its association to survival (196). Not all MMPs have purely tumor promoting effects. In mouse models MMP-8 has shown to suppress metastasis and, intriguingly, MMP-9 has been associated with tumor regression and anti-angiogenic activity in some, but not all, experimental models (197-199). Furthermore, MMP-9 was not elevated in a study by our group of human breast cancer compared to adjacent normal breast tissue in vivo, although MMP-1,-2,-3 were significantly elevated (35).

Urokinas plasminogen activator

The plasminogen activator system contributes to several physiological processes such as wound healing, tissue regeneration, angiogenesis and mammary gland development, but is also activated in pathological processes such as cancer progression and metastasis (200). Urokinas plasminogen activator (uPA) is one of two plasminogen activators and plays a crucial role in tumor invasion and metastasis. The uPA system consists of the serine protease uPA, a membrane anchored receptor and two inhibitors, serine proteases inhibitor-1 and -2 (PAI-1, PAI-2). uPA is activated by several proteases including plasmin and bound to its receptor uPA in turn activates plasmin in a reciprocal feedback loop. Plasmin plays a role in the degradation and remodeling of the basement membrane and the ECM as well as in the activation of growth factors and MMPs in malignancies. PAI-1 has an equivocal role in cancer with both an inhibitory effect on invasion and metastasis as well as facilitating tumor growth and dissemination (200, 201).

Urokinas plasminogen activator and breast cancer

uPA and PAI-1 have been confirmed and validated as prognostic biomarkers in breast cancer, especially in the node-negative subtype. High levels of uPA and, particularly PAI-1, have been associated with poor prognosis and have also shown to be predictive biomarkers for adjuvant chemotherapy (194, 202, 203).

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Angiogenesis

Angiogenesis, the formation of new blood vessels from an existing vascular network, is considered to be one of the hallmarks of cancer and is essential for tumor growth and metastasis (13). Angiogenesis begins via the activation of endothelial cells by proangiogenic factors such as VEGF, one of the key mediators of angiogenesis, and ILs. The VEGF family comprises five structurally related factors, of which VEGFA is the most important mediator of angiogenesis, and three receptors (204). It stimulates endothelial cell growth and facilitates the invasion of the underlying matrix by upregulation of specific proteases including pro-uPA and pro-MMPs, thereby leading to the formation of new blood vessels. VEGF plays an important role in embryonic development and wound healing in healthy individuals (205). Several mediators, including IL-1s, plasmin and MMPs, but also hypoxia induce VEGF gene expression in the ECM. Under physiological conditions these factors are produced by endothelial, stromal and blood cells, but during carcinogenesis the production is upregulated by tumor cells and the ECM. Hence, the levels of VEGF are elevated in several malignancies including colorectal, lung and breast cancer and correlates with metastatic spread (206). Preclinical studies have shown that a tumor cannot grow more than 1-2 mm3 without angiogenesis and that micro-metastases remain dormant without angiogenetic activity (207, 208). VEGF has been considered to be an ideal therapeutic target due to its crucial role in tumor growth and progression. Consequently, numerous clinical trials have been performed with anti-angiogenic pharmacological agents, including monoclonal antibodies and tyrosine kinase inhibitors. As of today eleven drugs have been approved for treatment of different malignancies. The clinical effects have been modest, probably due to acquired resistance to anti-angiogenetic therapy both by the tumor cells and the stromal cells (209).

Angiogenesis and breast cancer

Estrogen stimulates the secretion of VEGF in normal human breast tissue and it has been shown in several experimental breast cancer models that E2 stimulates VEGF and angiogenesis in vivo (210-212). Studies have shown an association between microvessel growth in the primary tumor and metastatic lesions in breast cancer supporting the role of angiogenesis in tumor progression and metastasis (213). The impact of angiogenesis on breast cancer development was also demonstrated in a meta-analysis showing an inverse association between tumor microvessel density (MVD) and survival of breast cancer patients (214). Furthermore, studies have shown that a high level of VEGF is a negative prognostic marker in breast cancer (215, 216).

Several anti-angiogenic drugs such as the monoclonal antibody bevacizumab, targeting VEGF, and several tyrosine kinase inhibitors, targeting growth factor receptors and

downstream signaling pathways, have been tested in numerous clinical trials for breast cancer, but the results have so far been disappointing with no or very limited effect on survival and as of today no anti-angiogenic drugs are considered to be standard treatment in breast cancer (217).